Breathing is the most fundamental motor behavior for terrestrial vertebrates. The frequency and amplitude of breathing movements are controlled by neural networks residing in the brainstem and spinal cord. In mammals, contraction of the diaphragm muscle is essential for driving airflow into the lungs during inspiration. Despite the complexity of the neural networks that regulate respiratory rhythms, diaphragm contraction is controlled by a single motor input, the activity of motor neurons (MNs) within the Phrenic Motor Column (PMC) in the cervical spinal cord. Loss of PMC neurons is the primary cause of death in degenerative MN diseases such as amyotrophic lateral sclerosis (ALS) and spinal cord injuries. Despite their essential role, the molecular determinants of PMC neuron identity are largely unknown. We have found that the development of PMC neurons requires the sustained activity of Hox5 transcription factors. Mice lacking Hox5 genes in MNs die of respiratory failure at birth and exhibit defects in multiple aspects of PMC identity, including cell body position, axon guidance and diaphragm innervation. In this proposal we will investigate the function of Hox5 genes in determining and maintaining phrenic MN identity. In Aim 1 we will determine temporally distinct functions of Hox5 proteins in phrenic MNs and how phrenic MN identity is maintained throughout lifetime. In Aim 2 we will define how Hox5 genes control phrenic MN specification at the transcriptional level. In Aim 3 we will identify direct Hox5 effectors and dissect their regulatory mechanisms. We have developed an integrative methodology encompassing genetic models, high-throughput sequencing, electrophysiology and behavioral assays, such as plethysmography, to address these questions in vivo. The overarching goal of this proposal is to uncover the basic principles underlying phrenic MN specification and maintenance so that we can begin to consider alternative treatment methods for respiratory dysfunction.